[Acute Eosinophilic Pneumonia (AEP) after Hookah Smoking]. / Akute eosinophile Pneumonie (AEP) nach Shisha-Rauchen.

A 27-year-old, previously physically healthy man presented to an emergency department with an acute onset of thoracic pain, dyspnea, non-productive cough and fever. Hours before the onset of symptoms, the patient has smoked tobacco using a waterpipe (spearmint taste, not commercially available in Germany). Due to a progressive respiratory failure the patient required invasive mechanical ventilation. The computertomography scan of the chest showed bilateral diffuse, infiltrative changes. The diagnosis of hookah smoking associated Acute Eosinophilic Pneumonia (AEP) was based on the patient's history, the eosinophilic count in broncho-alveolar lavage and the computertomographic findings. After treatment with corticosteroids, the patient could be extubated after 9 days. The outpatient follow-up revealed a normal lung function testing and X-ray of the chest without any physical sequelae. CONCLUSION: In cases of acute onset of thoracic pain, dyspnea, non-productive cough and fever, acute eosinophilic pneumonia should be considered for differential diagnosis in association with shisha smoking. Severe respiratory lung failure can be successfully treated with corticosteroid therapy and ventilation resulting in full recovery.

Human biological sample biobanking to support tissue biomarkers in pharmaceutical research and development.

Advances in the understanding of molecular pathology and thereby the mechanisms that could be amenable to therapeutic manipulation are the reason that pharmaceutical research and development is focused increasingly on measurement of molecular biomarkers in human biological samples. Obtaining direct or indirect access to sufficient samples that are fit for research purposes can be a major challenge. A biobanking infrastructure has a significant role in the acquisition, storage and usage of human biological samples and here we review some key requirements for establishing a biobank. These include ensuring; that appropriate governance mechanisms are in place, that samples available are appropriate and fit for the intended research purposes that the infrastructure is sustainable in the future and that use of the biobank assets meets the strategic aims of the host organisation. Finally we present a case study--the STRATUM project which has recently completed and through a collaborative approach involving six industry and public partners drawing on a network of experts, examined biobank policies, public attitudes to biobanking, donor consent, sample and data standards, technical requirements for a register and biobanking financial models, albeit from a UK perspective.

Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes.

Voltage-clamp analysis was applied to study the currents associated with the uptake of extracellular gamma-aminobutyric acid (GABA) by the cloned transporter GAT1 expressed at high efficiency in Xenopus oocytes. Steady-state GABA currents were increased at higher extracellular [GABA], [Na+], and [Cl-] and at more negative potentials. The Hill coefficient for Na+ exceeded unity, suggesting the involvement of two Na+ ions. In the absence of GABA, voltage jumps produced transient currents that behaved like capacitive charge movements; these were suppressed by the uptake inhibitor SKF-89976A, were shifted to more negative potentials at lower external [Na+] and [Cl-], and had an effective valence of 1.1 elementary charge. A turnover rate per transporter of 6-13/s at maximal [GABA] (-80 mV, 96 mM NaCl, 22 degrees C) is given both by the kinetics of voltage jump relaxations and by the ratio between the maximal GABA currents and the charge movements. These quantitative data are necessary for evaluating the roles of GAT1 in synaptic function.

Conducting states of a mammalian serotonin transporter.

We have studied permeation at a cloned rat 5-HT transporter expressed in Xenopus oocytes. [3H]5-HT uptake and [125I]RTI-55 binding yield a turnover rate of approximately 1/s that does not depend on membrane potential. However, in voltage-clamp experiments, three distinct currents results from 5-HT transporter expression. First, a steady-state, voltage-dependent transport-associated current is induced by 5-HT application. Second, a transient inward current is activated by voltage jumps to high negative potentials in the absence of 5-HT and is blocked by 5-HT itself. Third, a small leakage current is observed in the absence of 5-HT. All the observed currents are blocked by inhibitors of 5-HT uptake but are differentially affected by Na+, Li+, K+, Ba2+, Cs+, Cl-, and amiloride. The conducting states of the 5-HT transporter may reflect the existence of a permeation pathway similar to that of ionic channels.

Standard operating procedure for the collection of fresh frozen tissue samples.

Studies using fresh-frozen tissue samples originating from different centres, as is often the case in EORTC related translational research, can show conflicting research results due to heterogeneity in the quality of samples and associated data from each centre. The development of infrastructure for the European Human Frozen Tumour Tissue Bank (TuBaFrost) anticipated this problem and Standard Operating Procedures (SOPs) have been developed to ensure samples collected are of consistent high quality and variation in research results is minimised. The SOPs drew on the best practice standard workflows and operating procedures employed by members of the TuBaFrost Consortium and key tissue bank initiatives worldwide. It was essential to provide workable solutions that reflect the variety in infrastructure and resources at the potential collecting centres and also the fact that it is not necessary to standardise every step of the collection and storage process in order to collect high quality tissue. Hence, the TuBaFrost SOPs detail the compulsory measures that must be implemented in order to become a TuBaFrost collecting centre and also make advisory recommendations regarding the less critical factors. Accordingly, the TuBaFrost SOPs are very flexible and to illustrate this the complete SOP for collecting, freezing and storing tissue at the Erasmus MC Tissue Bank is included. These TuBaFrost SOPs could equally be applicable to centres collecting samples for EORTC related translational research studies in order to standardise sample quality and produce reliable and reproducible research results.

Topological localization of cysteine 74 in the GABA transporter, GAT1, and its importance in ion binding and permeation.

Xenopus oocytes expressing the GABA transporter GAT1 were exposed to membrane-impermeant sulfhydryl reagents, resulting in decreased GABA transport current, decreased capacitive charge movements, and increased Na+ and Li+ leakage currents. Mutation of cysteine 74 to alanine (C74A) eliminated these effects. The W68S and W68L mutations significantly increased and decreased the transporter's sensitivity, respectively, to sulfhydryl reagents. At each of the positions 73 through 76, cysteine residues were accessible to external MTSET. These findings, together with recent evidence placing the HD2-HD3 loop on the extracellular side, suggest that the HD2 region does not traverse the membrane.

Glutamate-101 is critical for the function of the sodium and chloride-coupled GABA transporter GAT-1.

We have investigated the possible role of selected negatively-charged amino acids of the sodium and chloride-coupled GABA transporter GAT-1 on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site-directed mutagenesis. The functional consequences were that one of the residues, glutamate-101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate-101 is critical for one or more of the conformational changes of GAT-1 during its transport cycle.

Measurement of transient currents from neurotransmitter transporters expressed in Xenopus oocytes.

Electrophysiological measurements add new dimensions to the study of neurotransmitter transporters. (1) One can perform measurements with high temporal resolution (however, uptake of radioactive substrate is limited in that it cannot resolve events that occur within 1 sec, which is greater than the time of a single transport cycle). (2) Electrophysiology provides information about partial steps in transport cycles, including the fact that ion binding and dissociation at transporters can generate currents, which provides new insights about ion-transporter interaction. (3) Electrophysiology provides information about single transporter molecules, from patch-clamp recordings of single-channel activity of neurotransmitter transporters. At present, little is known about the molecular mechanisms that underlie transport. Electrophysiological measurements of ion binding and permeation contribute to the analysis of mutations that affect transport. Electrophysiology may help to identify amino acids and domains in neurotransmitter transporters that participate in specific ways in the transport process, such as ion neurotransmitter binding, permeation pathways, voltage sensors, and gates. In combination with spectroscopic measurements, it may also be possible to identify the actual conformational changes of the proteins that enable substrate translocation.

Effect of Cs+, Li+ and Na+ on the potassium conductance and gating kinetics in the frog node of Ranvier.

The effects of monovalent internal cations Cs+, Li+ and Na+ on potassium channel conductance in the frog node of Ranvier were studied by means of the voltage clamp. As previously reported, when 10-80% of the internal K+ was replaced by one of the above cations, the steady-state current-voltage relationship was significantly modified. The main effect was a voltage-dependent attenuation of the currents. We demonstrate that the current attenuation is associated with a change in the channel gating kinetics. For small depolarizations the kinetics can be described by the usual potassium conductance activation time constant, tau n. However, under certain experimental conditions (e.g. substitution of the intracellular K+ with 10% Cs+), during larger depolarizations, stepping the membrane potential to values above 40-60 mV, the conductance develops with two time constants: tau n and a new, slower time constant that, in contrast to tau n, grows with membrane potential. These results can be explained by assuming that the cations may occupy two different sites in the channel; when the first site is occupied the channel is blocked, while occupation of the second site results in slowing of the gating kinetics in the affected channels.

Mechanism of hyperthyroidism-induced modulation of the L-type Ca2+ current in guinea pig ventricular myocytes.

The positive inotropic effects of thyroid hormone in the heart, increased force and velocity of contraction have been mostly attributed to modulation of myosin ATPase isoenzymes (V1, V2 and V3), and sarcoplasmic reticulum Ca2+ pumping activity. In addition, we have suggested that the effects on ventricular contraction result from a thyroid hormone-induced increase in L-type Ca2+ current (ICa,L). Due to the central role of ICa,L in excitation-contraction coupling, we studied mechanisms whereby thyroid hormone augments this current. Since thyroid hormone modulates adenylate cyclase activity in various tissues, we tested the hypothesis that the hormone activates adenylate cyclase, leading to increased cyclic adenosine monophosphate (cAMP) levels, protein kinase A activation, Ca2+ channel phosphorylation and increased ICa,L. We therefore stimulated or inhibited different sites along the "adenylate cyclase cascade", and measured ICa,L and isometric twitch in ventricular myocytes and papillary muscles from euthyroid and hyperthyroid guinea pigs. Our major findings were as follows. In euthyroid myocytes, 0.1 microM isoproterenol (Iso) increased ICa,L (at VM = 0 mV) from -7.04 +/- 0.72 to -22.26 +/- 1.88 pA/pF, P < 0.05, while in hyperthyroid myocytes (ICa,L = -21.48 +/- 2.94 pA/pF), Iso was ineffective. In euthyroid myocytes, intracellular application of cAMP (50 microM) was as potent as Iso, but ineffective in hyperthyroid myocytes. In hyperthyroid myocytes, a protein kinase A inhibitor (2 microM) lowered ICa,L from -26.82 +/- 1.54 to -10.17 +/- 1.70 pApF (P < 0.05), but had no effect in euthyroid myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and functional expression of a human Na(+) and Cl(-)-dependent neutral and cationic amino acid transporter B(0+).

A Na(+)-dependent neutral and cationic amino acid transport system (B(0+)) plays an important role in many cells and tissues; however, the molecular basis for this transport system is still unknown. To identify new transporters, the expressed sequence tag database was queried, and cDNA fragments with sequence similarity to the Na(+)/Cl(-)-dependent neurotransmitter transporter family were identified. Based on these sequences, rapid amplification of cDNA ends of human mammary gland cDNA was used to obtain a cDNA of 4.5 kilobases (kb). The open reading frame encodes a 642-amino acid protein named amino acid transporter B(0+). Human ATB(0+) (hATB(0+)) is a novel member of the Na(+)/Cl(-)-dependent neurotransmitter transporter family with the highest sequence similarity to the glycine and proline transporters. Northern blot analysis identified transcripts of approximately 4.5 kb and approximately 2 kb in the lung. Another tissue survey suggests expression in the trachea, salivary gland, mammary gland, stomach, and pituitary gland. Electrophysiology and radiolabeled amino acid uptake measurements were used to functionally characterize the transporter expressed in Xenopus oocytes. hATB(0+) was found to transport both neutral and cationic amino acids, with the highest affinity for hydrophobic amino acids and the lowest affinity for proline. Amino acid transport was Na(+) and Cl(-)-dependent and was attenuated in the presence of 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid, a system B(0+) inhibitor. These characteristics are consistent with system B(0+) amino acid transport. Thus, hATB(0+) is the first cloned B(0+) amino acid transporter.

H+ permeation and pH regulation at a mammalian serotonin transporter.

The rat serotonin transporter expressed in Xenopus oocytes displays an inward current in the absence of 5-HT when external pH is lowered to 6.5 or below. The new current differs from the leakage current described previously in two ways. (1) It is approximately 10-fold larger at pH 5 than the leakage current at pH 7.5 and reaches 1000 H+/sec per transporter at extremes of voltage and pH with no signs of saturation. (2) It is selective for H+ by reversal potential measurements. Similar H+-induced currents are also observed in several other ion-coupled transporters, including the GABA transporter, the dopamine transporter, and the Na+/glucose transporter. The high conductance and high selectivity of the H+-induced current suggest that protons may be conducted via a hydrogen-bonded chain (a "proton-wire mechanism") formed at least partially by side chains within the transporter. In addition, pH affects other conducting states of rat serotonin transporter. Acidic pH potentiates the 5-HT-induced, transport-associated current and inhibits the hyperpolarization-activated transient current. The dose-response relationships for these two effects suggest that two H+ binding sites, with pKa values close to 5.1 and close to 6.3, govern the potentiation of the 5-HT-induced current and the inhibition of the transient current, respectively. These results are important for developing structure-function models that explain permeation properties of neurotransmitter transporters.

Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation.

Single-channel activities were observed in outside-out patches excised from oocytes expressing a mammalian 5-hydroxytryptamine (5-HT) transporter. Channel conductance was larger for a mutant in which asparagine177 of the third putative transmembrane domain was replaced by glycine, suggesting that this residue lies within or near the permeation pathway. The N177G mutant enables quantitative single-channel measurements; it displays two conducting states. One state, with conductance of approximately 6 pS, is induced by 5-HT and is permeable to Na+. The other state (conductance of approximately 13 pS) is associated with substrate-independent leakage current and is permeable to both Na+ and Li+. Cl- is not a major current carrier. Channel lifetimes under all conditions measured are approximately 2.5 ms. The single-channel phenomena account for previously observed macroscopic electrophysiological phenomena, including 5-HT-induced transport-associated currents and substrate-independent leakage currents. The channel openings occur several orders of magnitude less frequently than would be expected if one such opening occurred for each transport cycle and therefore do not represent an obligatory step in transport. Nevertheless, single-channel events produced by neurotransmitter transporters indicate the functional and structural similarities between transporters and ion channels and provide a new tool, at single-molecule resolution, for detailed structure-function studies of transporters.

Nonsuture microsurgical vessel anastomosis.

An alternative microsurgical method for achieving vessel union without applying sutures is presented. The basic principles of the technique involve the creation of an extraluminal cuff that permits exact adaptation of both endothelial layers, without intraluminal damage or application of foreign material. When used for microsurgical procedures, the method can be further simplified by using a Teflon cuff secured by means of a mini-clip. The anastomosis can be accomplished quickly, in an uncomplicated and precise fashion. On the basis of preliminary experiments, this method appears to offer a wide range of applications, particularly if absorbable material is used.

Amino acid residues that control pH modulation of transport-associated current in mammalian serotonin transporters.

The rat and human serotonin transporters (rSERT and hSERT, respectively) were expressed in Xenopus oocytes and studied using site-directed mutagenesis, electrophysiological recordings, and [3H]5-HT uptake measurements. rSERT, but not hSERT, displayed increased transport-associated current at low pH. Chimeras and point mutations showed that, of the 52 nonidentical residues, a single residue at position 490 (threonine in rSERT and lysine in hSERT) governs this difference. Furthermore, potentiation required the glutamate residue at position 493. Cysteine substitution and alkylation experiments showed that residue 493 is extracellular. Cysteine at 493 increased, whereas aspartate decreased, the net charge movement per transported 5-HT molecule. The mutations at this region did not significantly affect other aspects of SERT function, including agonist-independent leakage current, voltage-dependent transient current, and H+ current. This region may therefore be part of an external gate required for rSERT function. The data and analyses show that, in the absence of detailed structural information, a gate-lumen-gate scheme is useful for interpreting results from mutations that alter functional properties of neurotransmitter transporters.

The effect of prostaglandin inhibitors on the laser-induced disruption of blood aqueous barrier in the rabbit.

In four series, each containing five pigmented rabbits, the therapeutic effects of different anti-inflammatory agents on the laser-induced disruption of the blood aqueous barrier was investigated. Utilizing an argon laser, the peripheral iris of the left eye of each animal was coagulated with a total energy input of 1,000 mJ. The right eyes served as controls. After the coagulation the intraocular pressure was monitored at intervals of 10 min, and the anterior chamber was tapped for analysis of the aqueous humour 100 min after treatment. In a fifth group of five rabbits, aqueous humour was analysed without laser or drug treatment. The protein concentration and the activity of the lactate dehydrogenase were analysed in all samples of aqueous humour. A significant effect on the protection of the blood aqueous barrier could be identified in the eyes pre-treated with indomethacin (2 mg/kg body weight, injected intramuscularly), and in those pre-treated with dexamethasone (12.8 mg/kg body weight, injected intravenously; P less than 0.025%). Pre-treatment with aspirin (20 mg/kg body weight, injected intravenously) also had a protective response in the eyes treated by laser. The significance of the results of clinical treatment is outlined.

Laser-produced lithium plasma as a narrow-band extended ultraviolet radiation source for photoelectron spectroscopy.

Extended ultraviolet (EUV) emission characteristics of a laser-produced lithium plasma are determined with regard to the requirements of x-ray photoelectron spectroscopy. The main features of interest are spectral distribution, photon flux, bandwidth, source size, and emission duration. Laser-produced lithium plasmas are characterized as emitters of intense narrow-band EUV radiation. It can be estimated that the lithium Lyman-alpha line emission in combination with an ellipsoidal silicon/molybdenum multilayer mirror is a suitable EUV source for an x-ray photoelectron spectroscopy microscope with a 50-meV energy resolution and a 10-mum lateral resolution.

The effect of topically applied prostaglandin inhibitors on the laser-induced disruption of the blood aqueous barrier.

The therapeutic effect of topically applied prostaglandin inhibitors on the laser-induced disruption of the blood-aqueous barrier was investigated in six series of five rabbits each. One series was not coagulated and served as baseline, and in a reference group laser coagulation was performed without pretreatment with a prostaglandin inhibitor. In four series the iris laser coagulation of the left eyes was preceded by topical application of a prostaglandin inhibitor. The right eyes served as controls for the contralateral effect on the blood aqueous barrier. After laser coagulation the intraocular pressure was monitored at 10-min intervals, and the anterior chamber was tapped for analysis of the protein concentration and the lactate dehydrogenase activity. Pretreatment with dexamethasone eyedrops and indomethacin eyedrops markedly blocked the laser-induced disruption of the blood-aqueous barrier. The level of protein concentration in the aqueous humor after laser coagulation was much less after pretreatment with dexamethasone or indomethacin eyedrops. The effect was significant, both for the laser-treated eyes and for the noncoagulated fellow eyes (p less than 0.025). The subconjunctival pretreatment with dexamethasone 1 or 24 h before laser coagulation had no significant effect with respect to the protection of the blood aqueous barrier.

A multi-substrate single-file model for ion-coupled transporters.

Ion-coupled transporters are simulated by a model that differs from contemporary alternating-access schemes. Beginning with concepts derived from multi-ion pores, the model assumes that substrates (both inorganic ions and small organic molecules) hop a) between the solutions and binding sites and b) between binding sites within a single-file pore. No two substrates can simultaneously occupy the same site. Rate constants for hopping can be increased both a) when substrates in two sites attract each other into a vacant site between them and b) when substrates in adjacent sites repel each other. Hopping rate constants for charged substrates are also modified by the membrane field. For a three-site model, simulated annealing yields parameters to fit steady-state measurements of flux coupling, transport-associated currents, and charge movements for the GABA transporter GAT1. The model then accounts for some GAT1 kinetic data as well. The model also yields parameters that describe the available data for the rat 5-HT transporter and for the rabbit Na(+)-glucose transporter. The simulations show that coupled fluxes and other aspects of ion transport can be explained by a model that includes local substrate-substrate interactions but no explicit global conformational changes.